1. Field of the Invention
This invention relates to diagnostic reagents which are useful in detecting specifically binding complexes in solutions and assay methods using such reagents. The specifically binding complexes, sometimes referred to as ligand-receptor complexes, include DNA/single-strand-DNA-binding-protein complexes, antigen/antibody complexes (such as DNA/anti-DNA complexes), and similar specifically binding complexes. Methods of labeling compounds for the direct determination of haptens such as biotin, into macromolecules is the subject of this application.
2. The Related Art
There are extensive teachings in the art of diagnostic assays and reagents involving specifically binding complexes. Antigen/antibody reactions are widely used to determine antigen and antibodies. The labeling of members of these complexes with detectable markers such as enzymes or florescent dyes is well known. The binding of antigen or antibodies to solid supports as a means of removing complexes from solutions is also known. The use of haptens such as biotin and anti-haptens such as streptavidin in diagnostic assay is extensively discussed in a review article that appears in Anal. Biochem., 171:1-32 (Wilchek & Bayer, 1988).
The extensive use of avidin/biotin technology in immunology and diagnostic medicine is based on numerous factors. The high binding affinity between avidin and biotin yields an avidin-biotin complex having very high stability. Avidin is a tetrameric glycoprotein made up of four identical subunits. Therefore, four biotin molecules are capable of binding to one avidin molecule, Adv. Prot. Chem. 29:85 (Green, N. M., 1975), thereby increasing the sensitivity of constructed probe systems, or allowing the crosslinking of 2 or more biotin-labeled materials.
The disassociation constant for the avidin-biotin complex is 10.sup.-15 at neutral pH, Biochem. J., 89:585 (Green, N. M., 1963). This interaction is one of the strongest non-covalent associations found in nature. Biotin is deeply bound in a groove in the avidin molecule with the carboxyl group about 9 angstroms below the protein surface, Biochem. J., 125:781 (Green, N. M., et al., 1971). The widespread use of biotin-avidin technology is based upon the fact that a biotin molecule is easily coupled via covalent linkage to a protein while maintaining substantially all the biological properties of the protein and also the binding capacity of biotin to avidin. However, steric hindrance can distort the biotin-labeled biomolecule when it is directly coupled to the biotin carboxyl group, causing decreased binding to avidin.
The introduction of an extended linking group can help eliminate steric impediments, Clin. Chem., 25: 1572 (Costello, S. M., et al., 1979), J. Immunol., 56:329 (Kentall, C., et al., 1983). Insertion of .epsilon.-aminocaproic acid (X-linker) as a spacer is one method of further separating the protein moiety of the biotinylated protein from the avidin molecule. One case where the insertion of an X-linker has been used is the study of biotinyl-insulin. Use of this X-linker between biotin analogs and insulin yielded complexes that were more stable than those lacking the spacer, Biochem. J., 21:978 (Hofmann, K., et al., 1982). Moreover, the use of an X-linker in biotinylated DNA probes enhanced the detectability of target DNA approximately fourfold. Proc. Natl. Acad. Sci., 80:4045 (Leary, J. J. 1983).
Biotin attached to a solid-support is described in Giese U.S. Pat. No. 4,282,287 with a continuation-in-part No. 4,478,914, (Giese) and a further continuation-in-part No. 4,656,252 (Giese). A precise layering technique wherein biotin is first attached to a solid-surface and the subsequent application of successive layers of avidin and extender, results in a controlled modification of surface characteristics.
European Patent No. 87,307,850.5 is directed toward a method for routine plant-virus diagnosis which includes biotin attached to a macro-molecule that is conjugated to a sample of probe DNA. The probe-containing-compound is applied to a solid-matrix which has a test sample of DNA derived from plant tissue immobilized thereon. The presence of the target sequence is determined by washing the matrix with enzyme-linked-avidin followed by assaying for enzyme activity associated with the matrix.
Galati et al. U.S. Pat. No. 4,467,031 described an enzyme-immunoassay which utilizes the biotin-avidin system as a convenient and stable linking group to connect a reporter enzyme to an antibody.
Hevey et al. U.S. Pat. No. 4,228,237 describes the use of the biotin-avidin system in a method for detection and determination of ligands. A surface having an antibody for the ligand of interest attached thereto is reacted with a sample of the ligand followed by a second-ligand-specific antibody that is conjugated with biotin. This coupling is then reacted with an avidin-conjugated-enzyme and results determined by measurement of enzyme activity.
Deutsch U.S. Pat. No. 4,656,025 describes a screening assay for tumor globulin. A tumor globulin-biotin conjugate on ELISA plates is reacted with avidin-conjugated-enzyme and quantification of the tumor globulin bound to the plate is determined by the application of the appropriate chromogenic substrate thereto.
Dressman et al. U.S. Pat. No. 4,535,057 describes an immunoassay having biotin conjugated to a solid support through an antibody-virus complex. This biotin-antibody virus complex is then reacted with avidin conjugated to a reporter group or a label and the presence of the label associated with the surface is indicative of the presence of virus in the sample.
Valkirs et al. U.S. Pat. No. 4,727,019, continuation-in-part of Valkirs et al. U.S. Pat. No. 4,632,901 describe an immunoassay wherein avidin is attached to a solid support and binds a ligand present in the sample to the support. U.S. Pat. No. 4,298,685 describes a diagnostic reagent that also involves avidin immobilized on a solid support. Rosentein U.S. Pat. No. 4,582,810 describes a detection system wherein a suspension of particles having avidin covalently bound thereto reacts with a biotin-antibody complex to form a still larger complex which results in a flocculent appearing solution.
Bacquet et al. U.S. Pat. No. 4,550,075 describes a method for ligand determination based on the biotin-avidin system without any solid support.
David et al. U.S. Pat. No. 4,486,530 describes an immunometric assay process that comprises a ternary complex of an antigenic substance and a first and second antibody bound to the antigen in which the complex is removed from solution by filtering through a membrane.
Clinical Chemistry 34, 8:1585 (Vilja et al. 1988) describes a monoclonal antibody based noncompetitive avidin-biotin assay for luteinizing hormone (LH) in urine.
Conventional methods for determining the incorporation of haptens into macromolecules involve assays such as the trinitrobenzene sulfonic acid (TNBS) assay (Snyder and Sobocinski, 1975, Analytical Biochemistry 64: 284-288) and assays based on ninhydrin (Samejima et al., 1971, Analytical Biochemistry 42: 237-247). The techniques involve the measurement of reactive groups present on the macromolecule of interest both before and after hapten or biotin derivatization. Incorporation of hapten or biotin is determined by difference. These conventional methods, however, are not very useful because they are destructive, inaccurate, and insensitive to the incorporation of small amounts of haptens, such as biotin, into large macromolecules.
Nucleic Acids Research 13: 745-761 (Forster et al., 1985) describes a photoactivatable biotin analogue, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3-pr opanediamine (photobiotin). This compound may be used for the preparation of large amounts of stable, non-radioactive, biotin-labeled DNA and RNA hybridization probes. Upon irridation with visible light, photobiotin forms stable linkages with nucleic acids. Hybridization complexes produced from photobiotin and a nucleic acid are detected by avidin-alkaline phosphatase or other similar reagents.
Biotechnology 5: 269-272 (McInnes et al., 1987) presents hybridization analyses using photobiotin-labeled single- and double-stranded DNA probes which demonstrate that such probes possess the sensitivity required to replace radioactive probes in routine experiments and diagnostic assays.